Phosphorylation-dephosphorylation of proteins is one of the most important mechanisms for the regulation of cellular functions. Protein kinase C (PKC) has emerged as a pivotal regulatory element for the regulation of many cellular functions. Multiple PKC subspecies have been identified by molecular cloning and the various isozymes were found to have distinct tissue, cellular, and subcellular distributions and were differentially expressed during development. The functional roles of these kinases in the control of cellular differentiation were tested with N1E115 neuroblastoma cells. PKC-activating phorbol ester counteracts the cAMP- induced neurite outgrowth of these cells by selective translocation and activation of PKC betaI but not PKCzeta. Activation of PKC betaI results in the phosphorylation of a M/r=90k protein, whereas activation of cAMP- dependent protein kinase attenuates its phosphorylation. A method for preparing substrates specific for a group of PKC isozymes has been devised based on onteraction of cationic polypeptide with Ca2+/phospholipid/detergent mixed micelles to form aggregates with a unique surface structure. Neurogranin (Ng), neuromodulin (Nm), and MARCKS are the most prominent PKC substrates in the brain. Ng and Nm bind calmodulin (CaM) with high affinity in the absence of Ca2+, whereas MARCKS does so in the presence of Ca2+. Phosphorylation of these proteins by PKC reduces their affinities toward CaM and thus release CaM for the CaM- dependent enzymes. In addition to the phosphorylation-induced effects, oxidation of Ng by peroxide or nitric oxide also reduces the affinity of this protein for CaM. Ng and Nm are dephosphorylated by protein phosphatase 1, 2A, and calcineurin and MARCKS is more effectively by protein phosphatase 1 compared to phosphatase 2A and calcineurin. These latter two phosphatases selectively recognize a set of sites in MARCKS, whereas phosphatase 1 dephosphorylates all the sites. Binding of phosphoinositides to MARCKS inhibits the phosphorylation of this protein. The phosphorylated MARCKS has a reduced affinity toward these phospholipids and may contribute to the translocation of this protein from membrane to cytosol upon stimulation of PKC. A novel M/r=28k PKC/casein kinase II substrate has been identified and its cDNA cloned. This protein has been shown to be a substrate of casein kinase II in N18 neuroblastoma cells. The genomic structures of the CNS-specific PKC gamma and Ng were analyzed for the purpose of defining tissue-specific and development- regulated expression of these two genes. The promoter activity of the Ng gene can be stimulated by phorbol ester or by co-transfection with PKC cDNAs. A M/r=20k protein binding to the AT-rich regions of both PKC gamma and Ng gene promoters has been identified and phosphorylation of this protein by PKC attenuates its binding to DNA elements containing ATTA, ATAA and AATA motifs.